Novel reagents for intracellular delivery of macromolecules

ABSTRACT

The present invention provides certain cationic lipids containing stigmasterol, ergosterol and cholic acid groups. Compounds of the invention are useful, either alone or in combination with other lipid aggregate-forming components (e.g., DOPE, DOSPA, DOTMA or cholesterol) for formulation into liposomes or other lipid aggregates. Such aggregates are cationic, and able to form stable complexes with anionic macromolecules, such as nucleic acids. The cationic lipids of the invention are useful in methods of transfecting cells, particularly to introduce nucleic acids into cells. The invention also related to kits for the preparation of lipid aggregates and to lipid aggregates and compositions for transfection of cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.09/326,106, filed Jun. 4, 1999, which is a continuation of U.S. patentapplication Ser. No. 08/195,866, filed Feb. 11, 1994, now U.S. Pat. No.6,075,012 and which is incorporated by reference in its entirety hereinto the extent not inconsistent herewith.

FIELD OF THE INVENTION

Cationic lipid compounds are disclosed, having utility in lipidaggregates for delivery of macromolecules and other compounds intocells. Also disclosed are compositions of cationic lipids and viralcomponents or non-viral fusagenic compounds useful for enhancingtransfection.

BACKGROUND OF THE INVENTION

Lipid aggregates such as liposomes have been found to be useful asagents for delivery to introduce macromolecules, such as DNA, RNA,protein, and small chemical compounds such as pharmaceuticals, to cells.In particular, lipid aggregates comprising cationic lipid componentshave been shown to be especially effective for delivering anionicmolecules to cells. In part, the effectiveness of cationic lipids isthought to result from enhanced affinity for cells, many of which bear anet negative charge. Also in part, the net positive charge on lipidaggregates comprising a cationic lipid enables the aggregate to bindpolyanions, such as nucleic acids. Lipid aggregates containing DNA areknown to be effective agents for efficient transfection of target cells.

The structure of various types of lipid aggregates varies, depending oncomposition and method of forming the aggregate. Such aggregates includeliposomes, unilamellar vesicles, multilameller vesicles, micelles andthe like, having particle sizes in the nanometer to micrometer range.Methods of making lipid aggregates are by now well-known in the art. Themain drawback to use of conventional phospholipid-containing liposomesfor delivery is that the material to be delivered must be encapsulatedand the liposome composition has a net negative charge which is notattracted to the negatively charged cell surface. By combining cationiclipid compounds with a phospholipid, positively charged vesicles andother types of lipid aggregates can bind DNA, which is negativelycharged, can be taken up by target cells, and can transfect targetcells. (Felgner, P. L. et al. (1987) Proc. Natl. Acad. Sci. USA84:7413-7417; Eppstein, D. et al., U.S. Pat. No. 4,897,355.)

Cationic lipids useful for transfection and intracellular delivery ofmacromolecules generally contain the following four structural elements:

The lipophilic group is a hydrophobic moiety which facilitates theinsertion of the cationic amphiphile into the membranes of the cell orliposome. The lipophilic group serves as an anchor for the cationicgroup (usually ammonium) which is positively charged at neutral Ph, toattach to the surface of the cell or liposome. The spacer arm istypically a hydrophilic, 2 to 15-atom moiety which connects the cationicgroup to the lipophilic group via the linker bond. The linker bond iseither an ether, ester, amide or other hydrolyzable bond.

A well-known cationic lipid disclosed in the prior art isN-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA).The structure of DOTMA is:

DOTMA by itself or in 1:1 combination withdioleoylphosphatidyl-ethanolamine (DOPE) is formulated into liposomesusing standard techniques. Felgner, et al. supra demonstrated that suchliposomes provided efficient delivery of nucleic acids to some types ofcells. A DOTMA:DOPE (1:1) formulation is sold under the trade nameLIPOFECTIN (Gibco/BRL: Life Technologies, Inc., Gaithersburg, Md.).Another commercially available cationic lipid is1,2-bis(oleoyloxy)-3-3-(trimethylammonia) propane (DOTAP), which differsfrom DOTMA only in that the oleoyl moieties are linked via ester, ratherthan ether bonds to the propylamine. DOTAP is believed to be morereadily degraded by target cells. A related group of prior art compoundsdiffer from DOTMA and DOTAP in that one of the methyl groups of thetrimethylammonium group is replaced by a hydroxyethyl group. Compoundsof this type are similar to the Rosenthal Inhibitor (RI) ofphospholipase A (Rosenthal, A. F. and Geyer, R. P. (1960) J. Biol. Chem.235:2202-2206) which has stearoyl esters linked to the propylamine core.The dioleoyl analogs of RI are commonly abbreviated as DORI-ether andDORI-ester, depending on the linkage of the fatty acid moieties to thepropylamine core. The hydroxy group can be used as a site for furtherfunctionalization, for example by esterification to carboxyspermine.

Another class of prior art compounds has been disclosed by Behr et al.(1989) Proc. Natl. Acad. Sci. USA 86:6982-6986; EPO publication 0 394111 (Oct. 24, 1990), in which carboxyspermine has been conjugated to twotypes of lipids. The structures of 5-carboxyspermylglycinedioctadecylamide (DOGS) is:

The structure of dipalmitoylphosphatidylethanolamine5-carboxy-spermylamide (DPPES) is:

Both DOGS and DPPES have been used to coat plasmids, forming a lipidaggregate complex that provides efficient transfection.

The compounds are claimed to be more efficient and less toxic than DOTMAfor transfection of some cell lines. DOGS is available commercially asTRANSFECTAM™ (Promega, Madison, Wis.).

A cationic cholesterol derivative (DC-Chol) has been synthesized andformulated into liposomes in combination with DOPE. (Gao, X. and Huang,L. (1991) Biochim. Biophys. Res. Comm. 179:280-285) The compound'sstructure is

Liposomes formulated with DC-Chol are said to provide more efficienttransfection and lower toxicity than DOTMA-containing liposomes for somecell lines.

Lipopolylysine, formed by conjugating polylysine to DOPE, has beenreported to be especially effective for transfection in the presence ofserum, a condition likely to be encountered in vivo (Zhou, X. et al.(1991) Biochim. Biophys. Acta 1065:8-14).

Despite advances in the field, a need remains for a variety of improvedcationic lipid compounds. In particular, no single cationic lipid todate has been found to work well with all cell types. Since differentcell types differ from one another in membrane composition, it is notsurprising that different compositions and types of lipid aggregates areeffective for different cell types, either for their ability to contactand fuse with target cell membranes, or for aspects of the transferprocess itself. Besides content and transfer, other factors are ofimportance, for example, ability to form lipid aggregates suited to theintended purpose, toxicity to the target cell, stability as a carrierfor the compound to be delivered, and ability to function in an in vivoenvironment. In addition, lipid aggregates can be improved by broadeningthe range of substances which can be delivered to cells. The cationiclipid compounds of the present invention have improved function withrespect to several of the foregoing attributes. Compositions of theinvention comprising cationic lipids in combination with viralcomponents or non-viral fusagenic compounds are particularly useful forenhancing the efficiency of transfection and/or the range of deliverycapabilities.

SUMMARY OF THE INVENTION

The present invention provides novel cationic lipids according to thegeneral formula:

In the general formula (I),

-   -   R is R_(A) or R_(B), where R_(A) is C₁₋₂₃ alkyl or alkenyl and        R_(B) is a steroid selected from the group consisting of        stigmasterol, ergosterol and cholic acid;    -   n is an integer ranging from 1 to about 2,000 where n is greater        than 1 only when Z is Z₁₆ or Z₁₇;    -   m is 0 or 1, where m is 0 when Z is Z₁₆ or Z₁₇ and m is 1 when Z        is Z₁-Z₁₅ or Z₁₈;    -   A is selected from any of A₁-A₃ where    -   A₁ is        -   where D_(A) and D_(B), independently of one another, are            selected from the group consisting of D₁-D₃ where:        -   D₁ is —Y₁—CO—Y₂—, where Y₁ and Y₂, independently of one            another, are O and N, wherein at least one of Y₁ and Y₂ is            N;        -   D₂ is —CH═CH—O—;        -   D₃ is —O— or —CO₂—; and        -   wherein E is selected from the group consisting of E₁-E₃            where:        -   E₁ is        -    where W₁ and W₂, independently of one another, are C₁₋₂₄            alkyl, alkenyl or aryl;        -   q is 1 to 6;        -   E₂ is —PO⁻ ₄—(CH₂)_(n)—NH—, where n is 2-6 with n of 2 being            preferred;        -   E₃ is —(PO⁻ ₄)_(r)-[inositol]-NH—, where r is 1 or 2;    -   A₂ is        -   where B_(A)-B_(C), independently of one another, are            selected from the group consisting of the following groups            B₁-B₄, wherein one of B_(A)-B_(C) is B₁, one of B_(A)-B_(C)            is B₂, and one of B_(A)-B_(C) is B₃ or B₄;        -   B₁ is —OH;        -   B₂ is —NH—R, where R is C₁₋₂₃ alkyl, alkenyl or acyl;        -   B₃ is —O-Z or —NH-Z; and        -   B₄ is —PO⁻ ₄—(CH₂)_(n)—NH-Z, where n is 2-6 with n of 2            being preferred;        -   A₃ is —NH—CH₂— or —CO—N—R₁—, where A is —NH—CH₂— when R is            cholic acid and A is —CO—N—R₁— when R is stigmasterol or            ergosterol;        -   where R₁ is an alkyl, alkenyl, alkynl, alkoxy, acyl or            alkylthio having from 1 to about 24 carbon atoms; and        -   where Z is selected from the group consisting of Z₁-Z₁₇            where        -   Z₁ is H except where W₁ and W₂ are methyl;        -   Z₂ is —(CH₂)_(n)—X, where n is 1-24 and X is selected from            the group consisting of Br, Cl, I and F;        -   Z₃ is —(CH₂)_(n)—NH₂, n=1-24;        -   Z₄ is —CH₂—NH— (CH₂)₃—NH— (CH₂)₄—NH₂;        -   Z₅ is —CH₂—NH— (CH₂)₃—NH— (CH₂)₃—NH— (CH₂)₃—NH₂;        -   Z₆ is —CH₂—NH—(CH₂)_(n)—NH₂, n=2-24;        -   Z₇ is -L-X where L is selected from the group consisting of            branched or straight chain alkyl, alkenyl, cycloalkyl, aryl,            alkoxy, thioalkyl and thioether groups having from 1 to            about 24 carbon atoms, and X is selected from the group            consisting of Br, Cl, I, F, NH₂ and [(NH₂)—(CH₂)_(n)]_(m)            where n is 2-24 and m is 1-24;        -   Z₈ is        -   Z₉ is        -    where n=1-24, D is H or other groups attached by amide or            alkyl amino groups;        -   Z₁₀ is a reporter molecule;        -   Z₁₁ is a protein, peptide or polypeptide;        -   Z₁₂ is a polysaccharide;        -   Z₁₃ is an amine or halide reactive group;        -   Z₁₄ is        -   Z₁₅ is        -    n=1-24, D is H or other groups attached by amide or alkyl            amino groups;        -   Z₁₆ is an amino acid;        -   Z₁₇ is a monosaccharide; and        -   Z₁₈ is a nucleic acid binding substance.

Compounds of the invention are useful, either alone or in combinationwith other lipid aggregate-forming components (e.g., DOPE, DOSPA, DOTMAor cholesterol) for formulation into liposomes or other lipidaggregates. Such aggregates are cationic, able to form stable complexeswith anionic macromolecules, such as nucleic acids. The lipid aggregatemacromolecular complex interacts with cells making the macromoleculeavailable for absorption and uptake by the cell. The halogenatedcompounds of the invention are also especially useful as intermediatesfor chemically coupling the cationic lipid to reporter molecules,proteins, polypeptides, antibodies, polysaccharides and the like topermit targeted delivery, quantitative assessment of targeting, greaterefficiency of delivery and enhanced range of delivery capabilities.

Compounds of the invention comprising a nucleic acid binding substanceare particularly useful in applications requiring targeted delivery toDNA or RNA. Nucleic acid binding substances include, without limitation,histones, protamines, polycationic peptides, intercalators, polyamines,and nucleic acid binding proteins or domains thereof. Useful nucleicacid binding proteins comprise DNA-binding motifs such as ahelix-turn-helix DNA binding motif or a zinc finger. Zinc fingers occurin a variety of eukaryotic transcription factors including Spl,estrogen, and glucocorticoid receptors, several Drosophila developmentalregulators, Xenopus Xfin protein, the E. coli UvrA protein, and certainretroviral nucleic acid binding proteins. Compounds of the inventioncomprising protein, peptide and polypeptide substituents are also usefulfor binding a variety of other substances. For example, lectins such asconcanavalin A and wheat germ agglutinin are useful for binding sugarsand β-N-acetylmuranlic acid and α-N-acetylneuraminic acid, respectively.

Compounds of the invention can conjugate to a variety of usefulmolecules and substances such as polyamines, polyamine acids,polypeptides, proteins, fluorescent dyes, intercalating dyes, reportermolecules, biotin, polysaccharides, monosaccharides, solid supportmaterials, magnetic beads, dendrimer particles, DEAE-Sephadex™(Pharmacia, Inc.), and the like. Depending on the specific compound ofthe invention and the substance to be conjugated thereto, conjugationcan occur using a compound of the invention as an alkylating agent,using a free amine thereof to react with an amine-reactive group of thesubstance to be conjugated, or by the use of cross-linking agents.

The present invention also provides compositions and methods fortransfecting eukaryotic cells comprising a cationic lipid and anenveloped virus, a component of an enveloped virus, or a fusagenicpeptide. Transfecting compositions comprise a cationic lipid compound ofthe invention in combination with an active or inactive enveloped virus,a viral component of an enveloped virus, or a non-viral fusagenicpeptide that functions to facilitate entry of cationic lipid aggregatesinto the cell. Transfection compositions also optionally contain agentswhich inhibit lysosomal enzymes or enhance release of material fromendosomes, such as chloroquine.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel cationic lipids having uniqueproperties and advantages not heretofore available to the liposome art.The compounds can be used alone or in combination with other compounds,for example, DOPE, to prepare liposomes and other lipid aggregatessuitable for transfection or delivery of compounds other than DNA totarget cells, either in vitro or in vivo.

Compounds of the invention having a halogen substituent (Z is Z₂ or Z₇)are additionally useful for synthesis of more complex cationic lipidshaving the halogen replaced by a desired compound. The convenience ofthe halogen as a useful leaving group makes such substitutionsstraightforward. Examples of useful substituents include, withoutlimitation, reporter groups, proteins, peptides, polypeptides,antibodies, carbohydrates, polysaccharides, and the like. Reportergroups can be any readily analyzed or visualized molecule, including,without limitation, fluorescent tags (Fluorescein, rhodamine),luminescent tags(4-methoxy-4-(3-phosphatephenyl)-spiro[1,2-di-oxetane-3,2′-adamantane](PPD)) biotin, dyes, chelators, affinity probes, etc. Such reportersenable visualization and measurement of target cell-lipid aggregateinteractions. Such reporters also provide a means for subsequentlyaccessing targeted cells, by providing surface binding sites unique totargeted cells. In addition, certain drugs and therapeutic compounds canbe substituted at the halogen site, by a metabolizable linkage, therebyenhancing efficiency of drug delivery. Also, DNA intercalating compoundsand nucleic acid binding substances can be substituted, providingfurther DNA binding and enhancing transfection efficiency. Compoundssuch as lectins can be substituted, thereby enhancing the range ofdelivery capabilities.

Compounds of the invention having ester-amide or amide linked lipophilicgroups are particularly useful for applications demanding metabolizable,less toxic compounds. Most of the less toxic lipids currently availablecontain ester linker bonds, which can be metabolized and catabolizedinto other lipid species in the treated cells. However, cationic lipidscontaining ester linker bonds are not stable when stored in an aqueoussolution. More stable compounds typically contain ether bonds which arenot readily hydrolyzable in the cell. The present invention resolves theproblems associated with prior art compounds by providing deliveryagents which are stable in aqueous solution, but are metabolizable andthus less toxic to the target cell.

Of those compounds in which the lipophilic group is attached viaester-amide or amide linker bonds, this invention includes, but is notlimited to, compounds represented by formulas II and III:

In formula II:

R₁ and R₂, independently of one another, are C₁₋₂₃ alkyl or alkenyl; Y₁and Y₂, independently of one another, are O and N, wherein at least oneof Y₁ and Y₂ is N; and W₁ and W₂, independently of one another, areC₁₋₂₄ branched or straight chain alkyl, alkenyl or aryl; q is 1 to 6;and Z is selected from the group consisting of Z₁-Z₁₃ and Z₁₈ where

-   -   Z₁ is H;    -   Z₂ is —(CH₂)_(n)—X, where n=1-24 and X is selected from the        group consisting of Br, Cl, I and F;    -   Z₃ is —(CH₂)_(n)—NH₂, n=1-24;    -   Z₄ is —CH₂—NH— (CH₂)₃—NH— (CH₂)₄—NH₂;    -   Z₅ is —CH₂—NH— (CH₂)₃—NH— (CH₂)₃—NH— (CH₂)₃—NH₂;    -   Z₆ is —CH₂—NH—(CH₂)_(n)—NH₂, n=2-24;    -   Z₇ is -L-X where L is selected from the group consisting of        branched or straight chain alkyl, alkenyl, cycloalkyl, aryl,        alkoxy, thioalkyl and thioether groups having from 1 to about 24        carbon atoms, and X is selected from the group consisting of Br,        Cl, I, F, NH₂ and [(NH₂)—(CH₂)_(n)]_(m) where n is 2-24 and m is        1-24;    -   Z₈ is    -   Z₉ is    -    where n=1-24, D is H or other groups attached by amide or alkyl        amino groups;    -   Z₁₀ is a reporter molecule;    -   Z₁₁ is a protein, peptide or polypeptide;    -   Z₁₂ is a polysaccharide;    -   Z₁₃ is an amine or halide reactive group; and    -   Z₁₈ is a nucleic acid binding substance.

Of particular interest are the products of Formula II in which W₁ and W₂are methyl groups and q is 1.

In formula III:

R₁, R₂, Y₁ and Y₂ are as defined above for Formula II, q is 2-6 with qof 2 being preferred; and Z is selected from the group consisting of Z₁,Z₃, Z₇, Z₁₀-Z₁₂, Z₁₄-Z₁₅, and Z₁₈ where

-   -   Z₁ is H,    -   Z₃ is —(CH₂)_(n)—NH₂, n=1-24,    -   Z₇ is -L-X where L is selected from the group consisting of        branched or straight chain alkyl, alkenyl, cycloalkyl, aryl,        alkoxy, thioalkyl and thioether groups having from 1 to about 24        carbon atoms, and X is selected from the group consisting of Br,        Cl, I, F, NH₂ and [(NH₂)—(CH₂)_(n)]_(m) where n is 2-24 and m is        1-24,    -   Z₁₀ is a reporter molecule,    -   Z₁₁ is a protein, peptide or polypeptide,    -   Z₁₂ is a polysaccharide,    -   Z₁₄ is    -   Z₁₅ is    -    n=1-24, D is H or other groups attached by amide or alkyl amino        groups, and    -   Z₁₈ is a nucleic acid binding substance.

Compounds of the invention having an enol-ether linked lipophilic groupare particularly useful in pH-controlled delivery of macromolecules. Theenol-ether compounds of the invention are susceptible to acid hydrolysisbut generally stable towards bases. Of those compounds in which thelipophilic group is attached via enol-ether linker bonds, this inventionincludes, but is not limited to, compounds represented by Formula IV:

In Formula IV:

R₁ and R₂, independently of one another, are C₁₋₂₃ alkyl or alkenyl;q=2-6 with q of 2 being preferred; and Z is selected from any of Z₁, Z₃,Z₇, Z₁₀-Z₁₂ and Z₁₄-Z₁₅, as defined above for Formula III.

Also included in this invention are compounds of formula I wherein thespacer arm comprises inositol or phosphoinositol. Compounds of theinvention having an inositol spacer arm are particularly useful ineffectively transfecting cells that contain receptors for inositolphosphates. Since phosphoinositides are found in both plants andanimals, the cationic analogues have a broad delivery range. Compoundscomprising an inositol or phosphoinositol spacer arm include, but arenot limited to, compounds represented by formula V:

In Formula V:

R₁ and R₂, independently of one another, are C₁₋₂₃ alkyl or alkenyl; A₁and A₂, independently of one another, are —O— or —CO₂—; n is 1 or 2,with n of 1 being preferable; and Z is selected from any of Z₁, Z₃, Z₇,Z₁₀-Z₁₂, Z₁₄-Z₁₅, and Z₁₈, as defined above for Formula III.

Additional subsets of compounds of formula I of this invention includecationic ceramides and sphingolipids. These compounds differ from thecationic lipids currently in use by lacking the glycerol(1,2-propanediol) spacer arm and the linker bond attaching thelipophilic moiety to the spacer arm is an alkyl bond, rather than aneasily hydrolyzable bond such as an ester or amide. Since ceramides andsphingolipids are present in nervous tissues, cationic analogues ofthese compounds are particularly useful for transfecting neural cells.The cationic ceramide and sphingolipid compounds of the inventioninclude, but are not limited to, compounds represented by formula VI:

In Formula VI:

R is a straight-chain or branched alkyl or alkenyl having one to about24 carbon atoms. Preferred R groups are 1-alkenyl having about 12 toabout 22 carbon atoms, with 1-pentadecene being most preferred.

A_(A)-A_(C), independently of one another, are selected from the groupconsisting of A₁-A₄, wherein one of A_(A)-A_(C) is A₁, one ofA_(A)-A_(C) is A₂, and one of A_(A)-A_(C) is A₃ or A₄, and wherein thegroups A₁-A₄ are as follows:

-   -   A₁ is OH;    -   A₂ is —NH—R, where R is C₁₋₂₃ alkyl, alkenyl or acyl;    -   A₃ is —O-Z or —NH-Z, where Z is selected from the group        consisting of Z₁-Z₁₃ as defined above for Formula II; and    -   A₄ is —PO⁻ ₄—(CH₂)_(n)—NH-Z, where n is 1-24, with n of 2 being        preferred, Z is selected from any of Z₁, Z₃, Z₇, Z₁₀-Z₁₂,        Z₁₄-Z₁₅ and Z₁₈, as defined above for Formula III.

Also included in this invention are compounds of Formula I wherein thelipophilic group is a steroid, rather than a long-chain hydrocarbon.Preferred steroids include stigmasterol, ergosterol and cholic acid.These compounds are useful in transfecting diverse types of cells.Cationic steroids of the invention include, but are not limited to,compounds represented by formula VII:A-B-Z  Formula VIIIn Formula VII:

A is a steroid selected from the group consisting of stigmasterol,ergosterol or cholic acid; B is —NH—CH₂— or —CO—N—R₁, where B is—NH—CH₂— when A is cholic acid and B is —CO—N—R₁— when A is stigmasterolor ergosterol; where R₁ is an alkyl, alkenyl, alkynyl, alkoxy, acyl oralkythio having from 1 to about 24 carbon atoms; and where Z is selectedfrom any of Z₃-Z₁₃ and Z₁₈ as defined above for general Formula I.

The compounds of the present invention also include those in which thecationic region is an amino acid which is indirectly or directlyattached, i.e. with or without a linker group, to the lipophilic moiety.These lipophilic polyamino acids are particularly useful forintracellular delivery of negatively charged macromolecules. This aspectof the invention is based on the premise that polycationic polyaminoacids alkylated with long hydrocarbon chains have enhanced affinity forcells, many of which bear a net negative charge, and for variouspolyanions, such as nucleic acids, relative to normal polyamino acids.Moreover, because of the relatively high lipid content of the alkylatedpolyamino acids, these compounds interact more strongly with the lipidbilayer of cell membranes than their cognate polyamino acids.

Lipophilic polyamino acids of the invention include, but are not limitedto, compounds represented by Formula VIII:

In Formula VIII:

Z₁ and Z₂, independently of one another, are amino acids selected fromthe group consisting of ornithine, lysine, arginine and histidine;

-   n is an integer (chain length) ranging from 1 to about 2,000 with n    of between 10 and 50 being preferred.

A₁ and A₂, independently of one another, are selected from the groupconsisting of the following groups X₁-X₆:

-   -   X₁ is a straight-chain alkyl, alkenyl, or alkynyl group having        from 2 to about 22 carbon atoms wherein one or more        non-neighboring —CH₂— groups can be replaced with an O or S        atom;    -   X₂ is a branched alkyl, alkenyl, or alkynyl group having from 2        to about 22 carbon atoms wherein one or more non-neighboring        —CH₂— groups can be replaced with an O or S atom;    -   X₃ is a straight-chain or branched alkyl group substituted with        one or two OH, SH, NH₂ or amine groups within about 3 carbon        atoms of the bond between X₃ and Z₁ or Z₂;    -   X₄ is a substituted straight-chain or branched alkyl, alkenyl or        alkynyl group having from 2 to about 22 carbon atoms wherein the        substituent is an aromatic, alicyclic, heterocyclic or        polycyclic ring and wherein one or more of the non-neighboring        —CH₂— groups of said alkyl, alkenyl or alkynyl group can be        substituted with an O or S atom.    -   X₅ is a —B-L group wherein B is selected from the group —CO—,        —CO₂—, —OCO—, —CO—N—, —O—CO—N—, —O—CH₂—, —CH₂—O—, —S—CH₂—,        —CH₂—S— or —CH₂— and L is selected from the group consisting of        -   X₁, X₂, X₄, or an aromatic, alicyclic, heterocyclic or            polycyclic ring moiety;    -   X₆ is a —CH(D-L)₂ or a —C(D-L)₃ group wherein D is selected from        the group consisting of —CO—, —CO₂—OCO—, —CO—N—, —O—CO—N—, —O—,        or —S— and L is selected from the group consisting of:        -   X₁, X₂, X₄, or an aromatic, alicyclic, heterocyclic or            polycyclic ring moiety.

Additional subsets of compounds of Formula I of this invention includethose in which the polycationic region is a modified polysaccharide. Thelipophilic polycationic polysaccharide compounds of the inventioncomprise a heteropolysaccharide or homopolysaccharide backbone,preferably a homopolysaccharide such as a glucan or galactan. Thepolysaccharide comprises between 2 and 2,000 monosaccharides, at leastone of which has a cationic substituent. Preferred substituents includetertiary amines, most preferably diethylaminoethyl. Each monomeric unithas between about 3 and about 7 carbon atoms, preferably 3 to 6, andmost preferably 6. Preferred polysaccharides include polymers ofmodified dextrans. Of particular interest are the products of generalFormula I in which Z is a modified glucose, m is 0, n is 50 to 600, andR is C₂₋₂₄ alkyl or alkenyl. Most preferred is lipophilic DEAE-dextran.Lipophilic polycationic polysaccharides show increased affinity fornegatively charged substances relative to the unsubstituted glycans.Moreover, because of the high lipid content, the modifiedpolysaccharides of the invention interact strongly with the lipidbilayer of cell membranes.

Lipophilic polycationic polysaccharides of the invention include, butare not limited to, compounds represented by Formula IX:

where Z₁ and Z₂, independently of one another, are monosaccharides,preferably glucose; n is an integer (chain length) ranging in value from1 to about 600, with n of between 50 and 200 being preferred; R₁ and R₂,independently of one another, are tertiary amines, preferablydiethylaminoethyl; and A₁ and A₂, independently of one another, areselected from the group consisting of groups X₁-X₆, as defined above forFormula VIII.

This invention also includes lipid aggregates comprising one or more ofthe compounds of Formulas II-IX or mixtures thereof. Of particularinterest are lipid aggregates of the compounds of Formulas II-IX whichmost closely simulate their naturally occurring counterparts.

The transfection methods of the present invention employing compounds offormulas II-IX or mixtures thereof can be applied to in vitro and invivo transfection of cells, particularly to transfection of eukaryoticcells including animal cells. The methods of this invention can be usedto generate transfected cells which express useful gene products. Themethods of this invention can also be employed as a step in theproduction of transgenic animals. The methods of this invention areuseful as a step in any therapeutic method requiring the introduction ofnucleic acids into cells. In particular, these methods are useful incancer treatment, in in vivo and ex vivo gene therapy, and in diagnosticmethods. The transfection compositions of this invention can be employedas research reagents in any transfection of cells done for researchpurposes. Nucleic acids that can be transfected by the methods of thisinvention include DNA and RNA from any source comprising natural basesor non-natural bases, and include those encoding and capable ofexpressing therapeutic or otherwise useful proteins in cells, thosewhich inhibit undesired expression of nucleic acids in cells, thosewhich inhibit undesired enzymatic activity or activate desired enzymes,those which catalyze reactions (Ribozymes), and those which function indiagnostic assays.

The compositions and methods provided herein can also be readily adaptedin view of the disclosure herein to introduce biologically activeanionic macromolecules other than nucleic acids including, among others,polyamines, polyamine acids, polypeptides, proteins, biotin, andpolysaccharides into cells. Other materials useful, for example astherapeutic agents, diagnostic materials and research reagents, can becomplexed by the cationic lipid aggregates and introduced into cells bythe methods of this invention.

The present invention also provides compositions and methods fortransfecting cells, preferably eukaryotic cells, comprising a cationiclipid and an enveloped virus, a component of an enveloped virus, amembrane virus, or a non-viral fusagenic compound. These transfectingcompositions comprise a cationic lipid compound of the invention incombination with an active or inactive enveloped virus, a viralcomponent of an enveloped virus, or a non-viral fusagenic peptide thatfunctions to facilitate entry of cationic lipid aggregates into thecell.

Viral components of enveloped viruses useful in transfectioncompositions include viral proteins, envelope fusion peptides,particular viral spike glycoproteins, multimers (i.e., dimers andtrimers) thereof, viral peptides of viral spike glycoproteins, and viralenvelope fragments containing embedded viral protein. The use of viralcomponents for enhanced transfection is exemplified with an inactiveSemliki Forest virus (SFV) particle in co-pending U.S. patentapplication Ser. No. 08/090,290 (filed Jul. 12, 1993), which isincorporated herein by reference in its entirety. Viruses with a broadhost range, such as alphaviruses, are generally preferred. However,viruses which exhibit cell specificity or a specific host range areparticularly useful for targeted delivery.

The mechanism of viral enhancement of cationic lipid/DNA complexdelivery is not fully understood. After the initial viral recognition ofthe host cell, certain viruses enter the cell by direct fusion with thecell membrane and, in certain cases, after neuramimidase cleavage ofpolysaccharides on the cell surface (influenza virus). The viral nucleicacid is then released into the cytoplasm. Other viruses enter the cellthrough endocytosis and are subsequently released from the endosomalcompartment. All steps are protein mediated. Although the mechanism isnot understood, these proteins, in conjunction with the cationiclipid/DNA complexes, enhance the uptake of macromolecules by the cell.

Inclusion of an enveloped virus, such as SFV, in a transfectioncomposition with cationic lipid aggregates complexed with nucleic acidsthus may enhance transfection compared to transfection mediated by thecationic lipid alone. Enhancement of transfection by alphaviruses,particularly SFV, is pronounced in historically hard-to-transfect celllines, including human primary cell lines. Enhancement of transfectionby enveloped viruses occurs in any cell which the virus can enter andinfect. Enhancement of transfection of alphaviruses, particularly SFV,occurs in cells which comprise cholesterol or another 3 β-OH-sterol intheir cell membrane. Methods for introducing a sterol or increasing thelevel of a sterol in cell membranes are known in the art and describedin the above-cited patent application (U.S. Ser. No. 08/090,290).

Non-viral fusagenic peptides (or membrane fusion proteins) have beenimplicated in cell fusion reactions. Although the mechanism is not fullyunderstood, fusagenic peptides can be added to transfection compositionscomprising cationic compounds of the present invention to enhance theefficiency of transfection.

The cationic lipids, viral or non-viral enhancing agents and nucleicacid of transfecting compositions can be combined in a variety of waysprior to contact with cells, as described, for example, in the citedcopending U.S. application. Transfection compositions also optionallycontain agents which inhibit lysosomal enzymes or enhance release ofmaterial from endosomes, such as chloroquine.

This invention also includes transfection kits which include one or moreof the compounds of formulas II-IX or mixtures thereof as cationiclipids. The invention also includes transfection kits comprising one ormore of the compounds of formulas II-IX (or mixtures thereof) incombination with a viral agent, a component of an enveloped virus, or anon-viral fusagenic peptide.

Definitions

Lipid Aggregate is a generic term which includes liposomes of all typesboth unilamellar and multilamellar as well as micelles and moreamorphous aggregates of cationic lipid or lipid mixed with amphiphaticlipids such as phospholipids and steroids.

Target Cell refers to any cell to which a desired compound is delivered,using a lipid aggregate as carrier for the desired compound.

Transfection is used herein to mean the delivery of expressible nucleicacid to a target cell, such that the target cell is rendered capable ofexpressing said nucleic acid. It will be understood that the term“nucleic acid” includes both DNA and RNA without regard to molecularweight, and the term “expression” means any manifestation of thefunctional presence of the nucleic acid within the cell including,without limitation, both transient expression and stable expression.

Delivery is used to denote a process by which a desired compound istransferred to a target cell such that the desired compound isultimately located inside the target cell or in, or on, the target cellmembrane. In many uses of the compounds of the invention, the desiredcompound is not readily taken up by the target cell and delivery vialipid aggregates is a means for getting the desired compound into thecell. In certain uses, especially under in vivo conditions, delivery toa specific target cell type is preferable and can be facilitated bycompounds of the invention.

Transfection-enhancing agent as used herein refers to any substancewhich, when used in conjunction with a cationic lipid, providessignificant enhancement of transfection (2-fold or more) overtransfection compositions comprising the cationic lipid alone.

Inactive virus refers to a virus which, after exposure to certainchemical or physical conditions, is no longer capable of expressing itsviral RNA. Viral inactivation is assessed by exposing viral particleswhose RNA contains a reporter gene under the control of the viralsubgenomic promoter to potentially inactivating conditions.

Non-viral fusagenic peptides (or membrane fusion proteins) refer toproteins or protein assemblies which interact with lipid components oftwo opposed bilayers of cell membranes so as to bring about theirunification. Although the mechanism is not fully understood, thefollowing non-viral fusagenic compounds have been implicated in cellfusion reactions: melitrin (the principal toxic peptide in bee venom),GALA (a synthetic peptide), snake venom cardiotoxins, snake venomcuraremimetic neurotoxins, myelin basic protein, bindin and lysin(proteins from abalone spermatozoa), prostaglandins, FUS1 and FUS2(products of the FUS1 and FUS2 genes of Saccharomyces cerevisiae), PH-30(a complex of two proteins involved in sperm/egg fusion), members of thecadherin family, and variants of the neural cell adhesion molecule(NCAM). Compounds involved in cell fusion reactions may be added tocompositions comprising compounds of the invention, thereby enhancingthe efficiency of transfection.

The cationic lipids were prepared by following the general reactionschemes given below (Schemes 1-11).

The quarternized ammonium/carbamate lipids are synthesized as shown inScheme 1. Alkyl isocyanate treatment of 3-dialkylamino-1,2-propanediolresults in the formation of compound 1. Compound 1 is then alkylatedwith methyl chloride or methyl iodide to obtain compound I (Formula II,Z=Z₁). Treatment of compound 1 with dihaloalkane results in compound II.Alkylation of compound 1 with bromoethylphthalamide followed byhydrazinolysis results in compound III (Formula II, Z=Z₃). CompoundsIV-VI are synthesized by treatment of compound II with the correspondingamine at high temperature. Compounds IX and X are synthesized bycondensing compound III with the corresponding amine-protected aminoacids and the subsequent removal of the protecting group. Compound XI issynthesized by treating compound III with reporter groups that containan activated carboxyl group.

The scheme provides a general method for the conjugation of lipids toany molecule or substance of interest. The alkyl halide II can be usedas a general alkylating agent. Thus, any molecule of interest that has anucleophilic moiety can react with compound II (Scheme 2) (J. March(1985) Advanced Organic Chemistry, John Wiley & Sons, New York, pp.364-366; Hilgetag & A. Martini, eds. (1972) Preparative OrganicChemistry, John Wiley & Sons, New York, pp. 448-460). Macromoleculesthat contain amino groups such as proteins and antibodies can beconjugated to lipids in this manner. Smaller molecules that containamino groups such as intercalators (methidium spermine), fluorescentdyes, nucleotides, nucleosides, amino acids, peptides and other reportermolecules such as biotin can also be conjugated in this manner.

Conversely, compounds III, IX-X, or IV-VI can be used for theconjugation of any molecules of interest that have electrophilic ornucleophilic sites. Compounds III, IX-X, or IV-VI can react withreporter molecules or other desired molecules if these molecules containcarboxylic acid sites, NHS ester or other active groups such asisothiocyanates, alkylhalides or chlorotriazines (Scheme 3) (Keezer, F.and Douraghi-Zdeh, K. (1967) Chem. Rev. 67:107; Dottario-Martin, B. andRavel, J. H. (1978) Anal. Biochem. 76:562; Staros, J. V. (1982)Biochemistry 21:3950.

Compounds III, IX-X, or IV-VI can also be conjugated with molecules thatcontain nucleophilic sites such as amines by using cross-linking agents(Scheme 4). Disuccinimidyl suberate can be used to conjugate compoundsIII, IX-X, or IV-VI to molecules that contain an amino group (Staros, J.V. (1982) Biochemistry 21:3990). Cross-linking agents that contain NHSester and maleimide can be used to conjugate compounds III, IX-X, orIV-VI to molecules that contain sulfhydryl group (Scheme 4) (Ji, T. H.(1979) Biochem. Biophys. Acta 559:39).

Phosphatidic carbamates (Formula III) are synthesized as shown in Scheme5. Compound 1 is treated with alkyl isocyanate to give compound 2.Removal of the BOC protecting group from compound 2 gives compound XV.Treatment of compound XV with bromoethylphthalamide followed withhydrazinolysis results in XVI. Compound XVII is obtained by treating XVIwith dihaloalkanes. Compounds XVIII and XIX are obtained by coupling XVIwith BOC-protected carboxy spermine or other amino acids, followed withthe removal of the protecting group. Compound XX is obtained by couplingXVI with proteins or peptides using cross linking agents.

Cationic analogs of sphingolipids wherein A_(A)-A_(C) are selected fromthe group consisting of A₁-A₃ (Formula VI) can be synthesized asdescribed in Scheme 6. Sphingosine (1) is alkylated to give compound 2.The primary hydroxyl group is selectively protected with dimethoxytrityl(DMTR) to obtain compound 3. Compound 3 is alkylated with bromoaceticacid in the presence of base to obtain compound 4. Treatment of compound4 with diamino alkane in the presence of DCC results in compound 5.

Compound 3 can be acylated with acetyl chloride to obtain compound 9which, on treatment with acid, yields compound 10. Compound 10 isalkylated with bromoacetic acid followed with treatment with DCC/diaminoalkane to obtain compound 12.

Compounds 5 and 12 can be acylated or alkylated as in Schemes 1-5 toobtain analogous compounds. Thus, treatment of compound 5 withBOC-protected carboxy spermine followed with treatment with acid resultsin the polycationic compound 6. Proteins or polypeptides can beconjugated to compounds 5, 8 or 12 using cross linking agents.

Cationic analogs of sphingolipids wherein one of AA-AC is A₄ (FormulaVI) can be synthesized as described in Scheme 7. Compound 1 isselectively acylated using N-hydroxysuccinimide (NHS) esters of fattyacids to obtain compound 2. Selective protection of the hydroxyl groupsusing dimethoxytritylchloride (DMTrCl) yields compound 3. DCC-mediatedcoupling of compound 3 with amine-protected aminoalkyl phosphate givescompound 4. Removal of the protecting groups yields compound 5. Compound5 can be converted to the various analogues using the methods describedabove in Schemes 3 and 4.

The cationic steroid compounds of the invention are synthesized as shownin Scheme 8. For example, cholic acid is treated with a diaminoalkane inthe presence of DCC to obtain compound 2. Compound 2 is then treatedwith BOC-protected carboxyspermine to give compound 3. Alternatively,compound 2 is conjugated to macromolecules such as proteins,polypeptides, polyamines, and polycationic compounds (see, e.g., groupR₃ in Scheme 2) by using a cross linking agent. Thus, compound 2 can betreated with cross linkers such as DSS followed with proteins orpolypeptides, for example, to obtain compound 4.

Compounds of the invention in which the lipophilic group is attached viaan enol-ether linker bond, such as compounds represented by formula IV,are prepared as shown in Scheme 9. Treatment of compound 2 with analdehyde (1) in the presence of acid followed with dehydration resultsin compound 3. The ester bond of compound 3 is hydrolyzed with base toobtain the corresponding alcohol 4. Compound 4 is then treated withcompound 1 in the presence of acid to obtain compound 5. Amine-protectedaminoalcohols (6) are conjugated to compound 5 using DCC as couplingagent to obtain compound 7. Removal of the protecting group fromcompound 7 yields compound 8. Compound 8 can be further modified asdescribed above for schemes 1-5.

The phosphoinositide lipids (Formula V) are synthesized as shown inScheme 10. The hydroxyl groups of inositol are protected using standardtechniques (see, e.g., J. Chem. Soc. (1987) 423-429) to producecompound 1. Compound 1 is then coupled to compound 2 using DCC as anactivator to obtain compound 3. The isopropylidene group of compound 3is removed with a mild acid to obtain compound 4. Compound 4 is thentreated with bromopropylphthalamide to obtain compound 5. Hydrazinolysisof compound 5 results in compound 6. Compound 6 can be further modified,as described above for schemes 1-5, to produce the various compounds offormula V. The phosphoinositide lipids thus can be conjugated to anymolecule or substance of interest, as exemplified with the carbamatelipids.

Lipophilic polyamino acids of the invention (Formula VIII) aresynthesized as shown, for example, in Scheme 11. Polylysine is partiallyacylated using compound 2 in the presence of a coupling agent (such asDCC) to produce the corresponding lipophilic polyamino acid. As will beappreciated by those skilled in the art, compound 2 can be anylipophilic moiety comprising an amine-reactive group. Alternatively, thepolyamino acid can be modified as described above for Schemes 1-5.

Lipophilic polycationic polysaccharides of the invention (Formula IX)can be similarly synthesized using standard coupling techniques asshown, for example, in Scheme 12. DEAE-dextran is partially acylatedwith R—CO—OH-DCC, where R is alkyl, alkenyl C₁₂₋₂₄, and treated withcompound 2 in the presence of DCC to obtain compound 3.

The compounds of the invention can be used in the same manner as areprior art compounds such as DOTMA, DOTAP, DOGS and the like. Methods forincorporating such cationic lipids into lipid aggregates are well-knownin the art. Representative methods are disclosed by Felgner et al.,supra; Eppstein et al. supra; Behr et al. supra; Bangham, A. et al.(1965) M. Mol. Biol. 23:238-252; Olson, F. et al. (1979) Biochim.Biophys. Acta 557:9-23; Szoka, F. et al. (1978) Proc. Natl. Acad. Sci.USA 75:4194-4198; Mayhew, E. et al. (1984) Biochim. Biophys. Acta775:169-175; Kim, S. et al. (1983) Biochim. Biophys. Acta 728:339-348;and Fukunaga, M. et al. (1984) Endocrinol. 115:757-761. Techniques forpreparing lipid aggregates of appropriate size for use as deliveryvehicles include sonication and freeze-thaw plus extrusion as perhapsthe most commonly used. See, e.g., Mayer, L. et al. (1986) Biochim.Biophys. Acta 858:161-168. Microfluidization is used when consistentlysmall (50-200 nm) and relatively uniform aggregates are desired (Mayhew,E., supra). Aggregates ranging from about 50 nm to about 200 nm diameterare preferred; however, both larger and smaller sized aggregates arefunctional.

Methods of transfection and delivery of other compounds are well-knownin the art. The compounds of the present invention yield lipidaggregates that can be used in the same processes as those prior artcompounds.

Viral components or non-viral fusagenic compounds can be combined withthe cationic lipids of the invention to enhance the efficiency oftransfection and/or the range of delivery capabilities. Methods forenhancing transfection using fusagenic compounds are well-known in theart. Preferred methods include those disclosed in co-pending U.S. patentapplication Ser. No. 08/090,290.

It will be readily apparent to those of ordinary skill in the art that anumber of general parameters are important for optimal efficiency oftransfection or delivery. These parameters include, for example, thecationic lipid concentration, the concentration of compound to bedelivered, the medium employed for delivery, the length of time thecells are incubated with the polyanion-lipid complex, and the relativeamounts of cationic and non-cationic lipid. It may be necessary tooptimize these parameters for each particular cell type. Suchoptimization is routine employing the guidance provided herein andknowledge generally available to the art.

It will also be apparent to those of ordinary skill in the art thatalternative methods, reagents, procedures and techniques other thanthose specifically detailed herein can be employed or readily adapted toproduce the liposomal precursors and transfection compositions of thisinvention. Such alternative methods, reagents, procedures and techniquesare within the spirit and scope of this invention.

1. A compound having the structure:

and salts thereof where: R is R_(A) or R_(B), where R_(A) is C₁₋₂₃ alkylor alkenyl and R_(B) is a steroid selected from the group consisting ofstigmasterol, ergosterol and cholic acid; m is 0 or 1, where m is 0 whenZ is Z₁₆ or Z₁₇ and m is 1 when Z is Z₁-Z₁₅ and Z₁₈; A is selected fromany of A₁-A₃ where: A₁ is

where DA is bonded to R and E is bonded to Z; R″, independently of R, isR_(A) or R_(B), where R_(A) is C₁₋₂₃ alkyl or alkenyl and R_(B) is asteroid selected from the group consisting of stigmasterol, ergosteroland cholic acid; D_(A) and D_(B), independently of one another, areselected from the group consisting of D₁-D₃ where: D₁ is —Y₁—CO—Y₂—,where Y₁ and Y₂, independently of one another, are O and NH, and whereinat least one of Y₁ and Y₂ is NH; D₂ is —CH═CH—O—; and D₃ is —O— or—CO₂—; E is selected from the group consisting of E₁-E₃ where E₁ is

 where W₁ and W₂, independently of one another, are C₁₋₂₄ alkyl, alkenylor aryl; q is 1 to 6; E₂ is —PO⁻ ₄—(CH₂)₂—NH—; and E₃ is —(PO⁻₄)_(r)[inositol]-NH—, where r is 1 or 2; A₂ is

where B_(A)-B_(C), independently of one another, are selected from thegroup consisting of the following groups B₁-B₄, wherein at least one ofB_(A)-B_(C) is B₁, at least one of B_(A)-B_(C) is B₂, and at least oneof B_(A)-B_(C) is B₃ or B₄, where: B₁ is —OH; B₂ is —NH—R, where R isC₁₋₂₃ alkyl, alkenyl or acyl; B₃ is —O-Z or —NH-Z; and B₄ is —PO⁻₄—(CH₂)₂—NH-Z; and A₃ is —NH—CH₂— or —CO—N—R₁—, where A is —NH—CH₂— whenR is cholic acid and A is —CO—N—R₁— when R is stigmasterol orergosterol; where R₁ is an alkyl, alkenyl, alkynl, alkoxy, acyl oralkylthio having from 1 to about 24 carbon atoms; and where Z isselected from the group consisting of Z₁-Z₁₅ or Z₁₈; where: Z₁ is Hexcept where W₁ and W₂ are methyl; Z₂ is —(CH₂)_(n)—X, where n is 1-24and X is selected from the group consisting of Br, Cl, I and F; Z₃ is—(CH₂)_(n)—NH₂, n=1-24; Z₄ is —CH₂—NH—(CH₂)₃—NH—(CH₂)₄—NH₂; Z₅ is—CH₂—NH—(CH₂)₃—NH—(CH₂)₃—NH—(CH₂)₃—NH₂; Z₆ is —CH₂—NH—(CH₂)_(n)—NH₂,n=2-24; Z₇ is -L-X where L is selected from the group consisting ofbranched or straight chain alkyl, alkenyl, cycloalkyl, aryl, alkoxy,thioalkyl and thioether groups having from 1 to about 24 carbon atoms,and X is selected from the group consisting of Br, Cl, I, F, NH₂ and[(NH₂)—(CH₂)_(n)]_(m) where n is 2-24 and m is 1-24; Z₈is

Z₉is

 where n=1-24, D is H or other groups attached by amide or alkyl aminogroups; Z₁₀ is a reporter molecule; Z₁₁ is a protein, peptide orpolypeptide; Z₁₂ is a polysaccharide; Z₁₃ is an amine or halide reactivegroup; Z₁₄ is

Z₁₅ is

n=1-24, D is H or other groups attached by amide or alkyl amino groups;and Z₁₈ is a nucleic acid binding substance.
 2. The compound of claim 1wherein A is A₃.
 3. The compound of claim 2 wherein A₃ is —CO—N—R₁— andR_(B) is stigmasterol.
 4. The compound of claim 2 wherein A₃ is—CO—N—R₁— and R_(B) is ergosterol.
 5. The compound of claim 1 wherein Dis D₃.
 6. The compound of claim 5 wherein R is R_(A), A is A₁ and E isE₃.
 7. A composition for transfecting a cell with a nucleic acid whichcomprise a nucleic acid and one or more compounds according to claim 1.8. A lipid aggregate which comprises one or more compounds of claim 1.9. A kit for preparing a lipid aggregate comprising one or more cationiclipids according to claim
 1. 10. A method for transfecting a cellcomprising the step of contacting the cell with a lipid aggregatecomprising a nucleic acid and a compound according to claim
 1. 11. Amethod for transfecting a cell comprising the step of contacting thecell with a lipid aggregate comprising a nucleic acid and one or morecationic lipids having the structure:

and salts thereof where: R is R_(A) or R_(B), where R_(A) is C₁₋₂₃ alkylor alkenyl and R_(B) is a steroid selected from the group consisting ofstigmasterol, ergosterol and cholic acid; m is 0 or 1, where m is 0 whenZ is Z₁₆ or Z₁₇ and m is 1 when Z is Z₁-Z₁₅ and Z₁₈; A is selected fromany of A₁-A₂ where: A₁ is

where D_(A) is bonded to R and E is bonded to Z; R″, independently of R,is R_(A) or R_(B), where R_(A) is C₁₋₂₃ alkyl or alkenyl and R_(B) is asteroid selected from the group consisting of stigmasterol, ergosteroland cholic acid; D_(A) and D_(B), independently of one another, areselected from the group consisting of D₁-D₂ where: D₁ is —Y₁—CO—Y₂—,where Y₁ and Y₂, independently of one another, are O and NH, and whereinat least one of Y₁ and Y₂ is NH; and D₂ is —CH═CH—O—; E is selected fromthe group consisting of E₁-E₃ where E₁ is

 where W₁ and W₂, independently of one another, are C₁₋₂₄ alkyl, alkenylor aryl; q is 1 to 6; E₂ is —PO⁻ ₄—(CH₂)₂—NH—; and E₃ is —(PO⁻₄)_(r)[inositol]-NH—, where r is 1 or 2; and A₂ is

 where B_(A)-B_(C), independently of one another, are selected from thegroup consisting of the following groups B₁-B₄, wherein at least one ofB_(A)-B_(C) is B₁, at least one of B_(A)-B_(C) is B₂, and at least oneof B_(A)-B_(C) is B₃ or B₄, where: B₁ is —OH; B₂ is —NH—R, where R isC₁₋₂₃ alkyl, alkenyl or acyl; B₃ is —O-Z or —NH-Z; and B₄ is —PO⁻₄—(CH₂)₂—NH-Z; and where Z is selected from the group consisting ofZ₁-Z₁₅ or Z₁₈; where: Z₁ is H except where W₁ and W₂ are methyl; Z₂ is—(CH₂)_(n)—X, where n is 1-24 and X is selected from the groupconsisting of Br, Cl, I and F; Z₃ is —(CH₂)_(n)—NH₂, n=1-24; Z₄ is—CH₂—NH—(CH₂)₃—NH—(CH₂)₄—NH₂; Z₅ is—CH₂—NH—(CH₂)₃—NH—(CH₂)₃—NH—(CH₂)₃—NH₂; Z₆ is —CH₂—NH—(CH₂)_(n)—NH₂,n=2-24; Z₇ is -L-X where L is selected from the group consisting ofbranched or straight chain alkyl, alkenyl, cycloalkyl, aryl, alkoxy,thioalkyl and thioether groups having from 1 to about 24 carbon atoms,and X is selected from the group consisting of Br, Cl, I, F, NH₂ and[(NH₂)—(CH₂)_(n)]_(m) where n is 2-24 and m is 1-24; Z₈ is

Z₉ is

 where n=1-24, D is H or other groups attached by amide or alkyl aminogroups; Z₁₀ is a reporter molecule; Z₁₁ is a protein, peptide orpolypeptide; Z₁₂ is a polysaccharide; Z₁₃ is an amine or halide reactivegroup; Z₁₄ is

Z₁₅ is

 where n=1-24, D is H or other groups attached by amide or alkyl aminogroups; and Z₁₈ is a nucleic acid binding substance.
 12. The method ofclaim 23 wherein in the cationic lipid when A is A₂, Z is Z₁₄.
 13. Acomposition for transfecting a cell with a nucleic acid which comprisesa cationic lipid of claim 1 capable of complexing said nucleic acid tobe transfected into said cell, and a transfection-enhancing agentselected from the group consisting of an enveloped virus, a membranevirus, a viral component, and a non-viral fusagenic compound.
 14. Acomposition according to claim 25 wherein said transfection-enhancingagent is an enveloped virus, and wherein said enveloped virus is analphavirus.
 15. A composition according to claim 26 wherein saidalphavirus is Semliki Forest virus.
 16. A composition according to claim25 wherein said transfection-enhancing agent is a viral component andwherein said viral component is selected from the group consisting ofviral proteins, envelope fusion peptides, viral spike glycoproteins,viral peptides of viral spike glycoproteins, and viral envelopefragments containing embedded viral protein.
 17. A composition accordingto claim 25 wherein said transfection-enhancing agent is a non-viralfusagenic peptide.
 18. A method for transfecting a cell comprising thesteps of contacting the cell with a transfecting composition of claim25.